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|Publication number
|Publication type
|Application number
|Publication date
|Apr 4, 2006
|Filing date
|Jun 9, 2003
|Priority date
|Feb 23, 2000
|Fee status
|Also published as
|Publication number
|Carl T. Brighton, Solomon R. Pollack
|Original Assignee
|The Trustees Of The University Of Pennsylvania
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|Patent Citations (28), Non-Patent Citations (33), Referenced by (38), Classifications (13), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The present patent application is a continuation-in-part patent application of U.S. patent application Ser. No. 10/257,126, filed Oct. 8, 2002, which is the U.S. national phase patent application of PCT/US01/05991, filed Feb. 23, 2001, which, in turn, claims the benefit of the filing date of U.S. Provisional Patent Application Ser. No. 60/184,491, filed Feb. 23, 2000.
The present invention is directed to the method of determining the voltage and current output required for the application of specific and selective electric and electromagnetic signals to diseased articular cartilage in the treatment of osteoarthritis, cartilage defects due to trauma or sports injuries, or as an adjunct with other therapies (e.g., cell transplantation, tissue-engineered scaffolds, growth factors, etc.) for treating cartilage defects in the human knee joint and a device for delivering such signals to a patient's knee.
The bioelectrical interactions and activity believed to be present in a variety of biological tissues and cells are one of the least understood of the physiological processes. However, there has recently been much research into these interactions and activity regarding the growth and repair of certain tissues and cells. In particular, there has been much research into stimulation by electric and electromagnetic fields and its effect on the growth and repair of bone and cartilage. Researchers believe that such research might be useful in the development of new treatments for a variety of medical problems.
Osteoarthritis, also known as degenerative joint disease, is characterized by degeneration of articular cartilage as well as proliferation and remodeling of subchondral bone. The usual symptoms are stiffness, limitation of motion, and pain. Osteoarthritis is the most common form of arthritis, and prevalence rates increase markedly with age. It has been shown that elderly patients with self-reported osteoarthritis visit doctors twice as frequently as their unaffected peers. Such patients also experience more days of restricted activity and bed confinement compared to others in their age group. In one study, the majority of symptomatic patients became significantly disabled during an 8-year follow-up period. Massardo et al., Ann Rheum Dis 48: 893–7 (1989).
Nonsteroidal anti-inflammatory drugs (NSAIDs) remain the primary treatment modality for osteoarthritis. It is unknown whether the efficacy of NSAIDs is dependent upon their analgesic or anti-inflammatory properties or the slowing of degenerative processes in the cartilage. There is also a concern that NSAIDs may be deleterious to patients. For example, NSAIDs have well known toxic effects in the stomach, gastrointestinal tract, liver and kidney. However, aspirin inhibits proteoglycan synthesis and normal cartilaginous repair processes in animals. One study in humans suggested that indomethacin might accelerate breakdown of hip cartilage. All adverse effects appear more commonly in the elderly—the very population the most susceptible to osteoarthritis.
In the disease commonly known as osteoporosis, bone demineralizes and becomes abnormally rarefied. Bone comprises an organic component of cells and matrix as well as an inorganic or mineral component. The cells and matrix comprise a framework of collagenous fibers that is impregnated with the mineral component of calcium phosphate (85%) and calcium carbonate (10%) that imparts rigidity to bone. While osteoporosis is generally thought as afflicting the elderly, certain types of osteoporosis may affect persons of all ages whose bones are not subject to functional stress. In such cases, patients may experience a significant loss of cortical and cancellous bone during prolonged periods of immobilization. Elderly patients are known to experience bone loss due to disuse when immobilized after fracture of a bone, which may ultimately lead to a secondary fracture in an already osteoporotic skeleton. Diminished bone density may lead to vertebrae collapse, fractures of hips, lower arms, wrists, ankles as well as incapacitating pains. Alternative nonsurgical therapies for such diseases are needed.
Pulsed electromagnetic fields (PEMF) and capacitive coupling (CC) have been used widely to treat nonhealing fractures and related problems in bone healing since approval by the Food and Drug Administration in 1979. The original basis for the trial of this form of therapy was the observation that physical stress on bone causes the appearance of tiny electric currents that, along with mechanical strain, were thought to be the mechanisms underlying transduction of the physical stresses into a signal that promotes bone formation. Along with direct electric field stimulation that was successful in the treatment of nonunion, noninvasive technologies using PEMF and capacitive coupling (where the electrodes are placed on the skin in the treatment zone) were also found to be effective. Pulsed electromagnetic fields generate small, induced currents (Faraday currents) in the highly conductive extracellular fluid, while capacitive coupling directly causes currents in the tissues; both PEMFs and CC thereby mimic endogeneous electrical currents.
The endogeneous electrical currents, originally thought to be due to phenomena occurring at the surface of crystals in the bone, have been shown to be due primarily to movement of fluid containing electrolytes in channels of the bone containing organic constituents with fixed negative charges, generating what are called "streaming potentials." Studies of electrical phenomena in cartilage have demonstrated a mechanical-electrical transduction mechanism that resembles those described in bone, appearing when cartilage is mechanically compressed, causing movement of fluid and electrolytes over the surface of fixed negative charges in the proteoglycans and collagen in the cartilage matrix. These streaming potentials apparently serve a purpose in cartilage similar to that in bone, and, along with mechanical strain, lead to signal transduction that is capable of stimulating chondrocyte synthesis of matrix components.
The main application of direct current, capacitive coupling, and PEMFs has been in orthopedics in healing of nonunion bone fractures (Brighton et al., J. Bone Joint Surg., 63: 2–13, 1981; Brighton and Pollack, J. Bone Joint Surg., 67: 577–585, 1985; Bassett et al., Crit. Rev. Biomed. Eng., 17: 451–529, 1989; Bassett et al., JAMA 247: 623–628, 1982. Clinical responses have been reported in avascular necrosis of hips in adults and Legg-Perthes's disease in children. Bassett et al., Clin. Orthop. 246: 172–176, 1989; Aaron et al., Clin. Orthop. 249: 209–218, 1989; Harrison et al., J. Pediatr. Orthop. 4: 579–584, 1984. It has also been shown that PEMFs (Mooney, Spine, 15: 708–712, 1990) and capacitive coupling (Goodwin, Brighton et al., Spine, 24: 1349–1356, 1999) can significantly increase the success rate of lumbar fusions. There are also reports of augmentation of peripheral nerve regeneration and function and promotion of angiogenesis. Bassett, Bioessays 6: 36–42, 1987. Patients with persistent rotator cuff tendonitis refractory to steroid injection and other conventional measures, showed significant benefit compared with placebo treated patients. Binder et al., Lancet 695–698, 1984. Finally, Brighton et al. have shown in rats the ability of an appropriate capacitive coupling electric field to both prevent and reverse vertebral osteoporosis in the lumbar spine (Brighton et al., J. Orthop. Res. 6: 676–684, 1988; Brighton et al., J. Bone Joint Surg., 71: 228–236, 1989).
More recently, research in this area has focused on the effects stimulation has on tissues and cells. For example, it has been conjectured that direct currents do not penetrate cellular membranes and that control is achieved via extracellular matrix differentiation (Grodzinsky, Crit. Rev. Biomed. Eng. 9:133, 1983). In contrast to direct currents, it has been reported that PEMFs can penetrate cell membranes and either stimulate them or directly affect intracellular organelles. An examination of the effect of PEMFs on extracellular matrices and in vivo endochondral ossification found increased synthesis of cartilage molecules and maturation of bone trabeculae (Aaron et al., J. Bone Miner. Res. 4: 227–233, 1989). More recently, Lorich, Brighton et al. reported (Clin. Orthop. Related Res. 350: 246–256, 1998) that signal transduction of a capacitively coupled electric signal is via voltage gated calcium channels, leading to an increase in cytosolic calcium with a subsequent increase in activated (cytoskeletal) calmodulin.
Much research has been directed at studying tissue culture in order to understand the mechanisms of response. In one study, it was found that electric fields increased [3H]-thymidine incorporation into the DNA of chondrocytes, supporting the notion that Na+ and Ca2+ fluxes generated by electrical stimulation trigger DNA synthesis (Rodan et al., Science 199: 690–692, 1978). Studies have found changes in the second messenger, cAMP, and cytoskeletal rearrangements due to electrical perturbations (Ryaby et al., Trans. BRAGS 6: 1986; Jones et al., Trans. BRAGS 6: 51, 1986; Brighton and Townsend, J. Orthop. Res. 6: 552–558, 1988). Other studies have found effects on glycosaminoglycan, sulfation, hyaluronic acid, lysozyme activity and polypeptide sequences (Norton et al., J. Orthop. Res. 6: 685–689, 1988; Goodman et al., Proc. Natn. Acad. Sci. USA 85: 3928–3932 1988).
It was reported in 1996 by the present inventor that a cyclic biaxial 0.17% mechanical strain produces a significant increase in TGF-β1 mRNA in cultured MC3T3-E1 bone cells (Brighton et al., Biochem. Biophys. Res. Commun. 229: 449–453, 1996). Several significant studies followed in 1997. In one study it was reported that the same cyclic biaxial 0.17% mechanical strain produced a significant increase in PDGF-A mRNA in similar bone cells (Brighton et al., Biochem. Biophys. Res. Commun. 43: 339–346, 1997). It was also reported that a 60 kHz capacitively coupled electric field of 20 mV/cm produced a significant increase in TGF-β1 in similar bone cells (Brighton et al., Biochem. Biophys. Res. Commun. 237: 225–229, 1997). However, the effect such a field would have on other genes has not been reported in the literature.
In the above-referenced parent patent application, entitled "Regulation of Genes Via Application of Specific and Selective Electrical and Electromagnetic Signals," methods were disclosed for determining the specific and selective electrical and electromagnetic signals for use in creating specific and selective fields for regulating target genes of diseased or injured tissues. The present invention builds upon the technique described therein by describing the method of determining the voltage and current output required and the corresponding apparatus for delivering specific and selective electrical and electromagnetic signals to the human knee joints in patients afflicted with osteoarthritis and other cartilage defects, diseases and injuries.
The present invention relates to treating osteoarthritis and other cartilage diseases, defects, and injuries in human knee joints via the application of specific and selective fields generated by specific and selective electric and/or electromagnetic signals. The invention includes a method of determining the voltage and current of the signal to apply to electrodes or coils applied to the knee for treatment.
More particularly, the invention relates to a method of treating diseased tissue in a human through the application of a specific and selective electric or electromagnetic field to diseased tissue in a human, including osteoarthritis and other cartilage diseases, defects and injuries in the knee. The method includes the steps of determining the voltage and current output that provides the treatment of diseased tissue in an animal model corresponding to the diseased tissue of the human, determining the anatomic dimensions and total tissue volume of the diseased tissue in the animal model, determining the anatomic dimensions of total tissue volume of the diseased tissue of the human, scaling of the voltage and current output used in the animal model based on the comparative anatomical dimensions and the total tissue volume of the diseased tissue of the human to the total tissue volume of the diseased tissue in the animal model, and applying the scaled voltage and current to the diseased tissue of the human.
In order to determine the anatomic and tissue volume factors of the human knee, an analytical model of the human knee was developed that accounts for the contributions to total tissue volume by the different components of the knee joint. The conductivities of the tissues between electrodes applied to the knee, the electric field amplitude for bone, cartilage, marrow, muscle and fat in the knee, the current density amplitude for bone, cartilage, marrow, muscle and fat in the knee, the electric field amplitude for different thicknesses of articular cartilage in the knee, the electric field amplitude for knees of different sizes, the electric field amplitude without subcutaneous fat in the knee, the current density amplitude with and without subcutaneous fat in the knee, the electric field amplitude and current density for at least two longitudinal positions of the electrodes relative to the knee, and variations of at least two effective driving signals applied to the body from an external signal generator are determined from derived electric field and/or current density histograms for a determination of the current and voltage signals to apply to the knee to create the desired therapeutic field.
It is understood that a single value of applied voltage, at a particular frequency, across an anatomical structure such as a knee results in a range of values of electric field and current density in each tissue compartment. This results from the spatial complexity of anatomical compartments in living structures and their different electrical properties. Accordingly, the histograms derived by this analysis presents the percent of tissue volume for which the electric field or current density has a specific value as a function of the range of electric field and current density values. For example, the application of a sine value voltage of 0.25 V amplitude at 60 kHz results in a range of peak values in the cartilage/synovium, of the electric field amplitude of approximately 8 mV/cm to 200 mV/cm in a knee the size of a rabbit knee while in a human knee a sine wave voltage amplitude of 5 V @ 60 kHz would be required to achieve the approximate overlapping range of peak values. The range of values of the electric field amplitude in the treated tissue is an important "dose" parameter in this invention.
The range of values obtained from the analytical model must encompass the values obtained from detailed studies of cellular responses to the stimulating electrical fields. One therefore measures the electrical field dose-response of cells taken from the target tissue to determine the field amplitudes that are most effective. Then, from the histograms shown, one determines the external voltage and currents to apply, first to animal models and then to humans, in order to encompass these effective electrical parameters at the tissue targeted for healing. In the example set forth here, a frequency of 60 kHz for the applied signal is used to obtain specific numerical values. Accordingly, the impedance values of all tissue compartments were taken at the frequency of 60 kHz. One knowledgeable in the field could perform the same analysis at other frequencies, adjust the tissue impedances to their values at the new frequency and obtain different values for the ranges of the electrical field and current density at any chosen frequency or set of frequencies.
The invention also includes a method and a device for treating diseased tissue (such as osteoarthritis), defective or injured tissue in a human knee joint through the application of a specific and selective electric or electromagnetic field to the afflicted tissue in the human knee joint. Such a device in accordance with a capacitive coupling embodiment of the invention includes at least two electrodes adapted for application in the proximity of a patient's knee joint and a signal generator that generates electric signals for application to the electrodes so as to produce an electric field of amplitude approximately 8 mV/cm to 360 mV/cm and a current density from approximately 8 μA/cm2 to 300 μA/cm2 within the synovium and articular cartilage of the patient's knee joint. An inductive coupling embodiment of the invention includes a coil adapted and configured to receive the electric signals to produce these electric fields. Preferably, the signal generator provides one of a plurality of output electric signals with a voltage selected by a user in accordance with a size of the human knee joint. Larger knee joints receive signals with larger voltages.
These and other aspects of the present invention will be elucidated in the following detailed description of the invention.
The present invention will be apparent from the following detailed description of the invention taken in conjunction with the accompanying drawings, of which:
The invention will be described in detail below with reference to
As used herein, the phrase "signal" is used to refer to a variety of signals including mechanical signals, ultrasound signals, electromagnetic signals and electric signals output by a device.
As used herein, the term "field" refers to an electrical field within targeted tissue, whether it is a combined field or a pulsed electromagnetic field or generated by direct current, capacitive coupling or inductive coupling.
Previous studies by the present inventors have shown that a capacitively coupled field significantly increased the proliferation of bone cells grown in culture (Brighton, Pollack, et al., J. Orthop. Res. 3: 331–340, 1985) and significantly increased the rate of healing in a rat fractured fibula model (Brighton, Pollack, et al., Clin. Orthop. Related Res. 285: 255–262, 1992). Also, the field distributions in the vertebral bodies of rats during capacitively coupled electrical stimulation have been determined (Carter, Vresilovic, Pollack, and Brighton, IEEE Trans. Biomed. Eng. 36(-3): 333–334, 1989). In order to determine the required output voltage and current required to produce an equivalent electric field and current density in a human knee as found in bone cell and rabbit fibula studies, the analytical model depicted in
As shown in
In the analytical model of
Material Complex Conductivity (S/m) at 60 kHz Muscle Parallel to fibers 7.0 � 10−1 Perpendicular to fibers 2.0 � 10−1 Marrow 2.0 � 10−1 Bone Cortex 1.0 � 10−2 Cartilage 8.9 � 10−2 Subcutaneous Fat 2.0 � 10−1 Admittance/Area (S/cm2) at 60 kHz Electrode-Dermal 3.0 � 10−3 Interface
As will be explained below, the cartilage and synovial fluid compartment 5 was computed for three sizes: 0.3 cm, 2.3 cm, and 4.3 cm, and the applied electrode potential (potential between the two electrodes) was taken as 1.0 V.
The approximate results for the analytic model of the human knee for an applied voltage of 1.0 V peak-to-peak at 60 kHz are given below for various parameters as a function of the electric field and the current density.
As shown in
As shown in
As shown in
The amount of subcutaneous fat has also been found to have little effect on field and current density. Subcutaneous fat has a lower conductivity than muscle and, therefore, the electric field (
Finally, it has been discovered that the effect of improper alignment of the electrodes 1 placed on the knee relative to the cartilage/synovial compartment 5 is not critical. Four cases were considered: 1) mid-gap (middle of the cartilage/synovial compartment 5); 2) half diameter below the gap; 3) diameter below the gap; and 4) 2 diameters below the gap. The results for the electric field are shown in
Previous work by the present inventors on stimulation of fracture healing with capacitive coupling led to the following understanding of field and current levels as a progression was made sequentially from bone cell studies to animal studies to human studies:
Cell (in vitro)
Maximum effect at 20 mV/cm electric field
Current density of 200 μA/cm2.
Animal (rabbit)
Voltage output of 0.25 Vp-p for the device with
Results: Successful
electrodes on the rabbit knee. Approximate range
healing of Fracture
of peak of the electric field of 8–200 mV/cm in the
fracture callus. Approximate range of peak
electrical current density 8–195 μA/cm2 in the
fracture callus.
Human (nonunion)
Voltage output of 5 Vp-p for the device with the
Results: Successful
electrodes on the skin. Approximate range of peak
healing Nonunion
electric field of 8–360 mV/cm. Electric current
density of 8–300 μA/cm2
This is shown in
As a result of these prior comparisons, the present inventors have determined the following therapeutic effective ranges of field (E) and current density in the callus (see
(J) Animal/Human Callus Bone Cells E 8 mV/cm to 180 mV/cm 20 mV/cm J 8 μA/cm2 to 180 μA/cm2 200 μA/cm
These animal/human ranges of electrical field amplitude and current density take an important factor into account. It is that their extreme limits must encompass the results obtained from bone cell studies where geometric factors permitted more exact field determinations. In this way regions of the human/animal tissue cycle through the therapeutic effective range of the electric field and current density as the sinusoidal applied voltage cycles through the sine wave.
Since in vitro cartilage cell results also occurred at an effective field of 20 mV/cm, and since the measured circumference of the human knee joint is approximately the same as the measured diameter of the mid-calf region of the leg in a given patient, then the same scale factor of 5, used in the human tibia fracture model (
Thus, if the circumference of a given human knee=15.7 inches, then the diameter=5 inches. This was found to be the most common mean knee circumference measured in patients with osteoarthritis. An output voltage of 5 Vp-p should produce the above E field and current density in the average size knee joint cartilage/synovial compartment. A smaller knee joint diameter, say 4.6 inches as an example, would require less voltage output (4.6 Vp-p), and a very large knee joint diameter, say 7.6 inches, would require more voltage output (7.6 Vp-p). Thus, a voltage output of 4.6 Vp-p to 7.6 Vp-p would cover the full range of knee sizes measured to date to produce the desired therapeutic E field and current density in the cartilage/synovial fluid compartment of the knees as described above. Variations of �10% in the applied voltage are tolerated because of the distribution of values E and J resulting from anatomical complexity.
Thus, in accordance with the invention, the approximate size (and hence approximate diameter) of the patient's knee is determined, and a signal is generated and applied to the electrodes that will generate the desired electric field in the range of 8 mV/cm to 360 mV/cm and a current density range of 8 μA/cm2 to 300 μA/cm2 for treatment of osteoarthritis in the knee, for example. Preferably, the signal generator includes a select control (
Also in accordance with the invention, the appropriate electric field can be delivered to diseased or traumatized articular cartilage using an inductive coupling device of the type shown in
Schematic diagrams showing a coil of N total turns of wire and a cross-section through this coil are shown in
and I is the current in the coil, l is the length of the coil and μ0 is the magnetic permeability of air (and tissue). In the central region of the coil, the field of a solenoid is quite uniform so we can select the values of l1 and l2 so that they are equal. Since sinθ1 is a negative number because l1 is negative along the Z axis, sin θ2−sin θ1=2 sin θ2 if l1=l2.
Therefore, Equation (1) becomes:
The current, I is a sinusoidal varying current and it can be written as:
I=I 0 −e iωt (3)
where I0 is the amplitude of the sinusoidal current, ω is 2π times the frequency (which we will set to 60 kHz, and i is the imaginary number √{square root over (−1)}. This notation is equivalent to the following:
I=I0 sin ωt (4)
Therefore, Equation (2) becomes:
From Maxwell's Equations we obtain an expression for the amplitudes of the electric field associated with the time-varying magnetic flux. It should be noted that the magnetic flux is parallel to the long axis of the leg and is therefore perpendicular to the planes of the cartilage in the knee. The electric field, however, is in the planes of the cartilage, is circular in direction, and will vary in amplitude with radius such that at the geometric center of the knee, the electric field amplitude will be zero and it will increase in amplitude in a linear dependence on radius.
The theory says that
where |E| is the electric field amplitude, dB/dt is the time derivative of Equation (5), and r is the distance from the geometric center of the knee joint to an arbitrary radius, r. Placing Equation (5) into equation (6) and performing differentiation gives
where the cos ω indicates that the electric field is 90� out of phase with the magnetic flux.
If we insert the values of all the parameters in Equation (7), namely
where |E|Max is the amplitude at the maximum radius of the knee, namely 6.35�10−2 meters.
Therefore, for any given cycle of the magnetic field, the center of the knee has zero electric field, while at the extreme radius, the field cycles from 0 to 315 mV/cm. For 90% of the area of the cartilage synovial region to achieve an electric field strength of 20 mV/cm, the maximum field at the periphery of the knee joint must be 65 mV/cm. This means that the number of turns (N) in the transducer can be reduced from 3000 to (65/315)�3000=620.
Although implementations of the invention have been described in detail above, those skilled in the art will readily appreciate that many additional modifications are possible without materially departing from the novel teachings and advantages of the invention. Any such modifications are intended to be included within the scope of the invention as defined in the following claims.
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text:
Page 1
Elf5 Regulates Mammary Gland Stem/Progenitor Cell Fate by
Influencing Notch Signaling
RUMELA CHAKRABARTI,a,bYONG WEI,bROSE-ANNE ROMANO,aCHRISTINA DECOSTE,bYIBIN KANG,bSATRAJIT SINHAa
aDepartment of Biochemistry, Center of Excellence in Bioinformatics and Life Sciences, State University of New
York at Buffalo, Buffalo, New York, USA;bDepartment of Molecular Biology, Princeton University, Princeton,
New Jersey, USA
Key Words. Elf5•Mammary gland•Alveologenesis•Mammary stem cells•Notch•Knockout
The transcription factor E74-like factor 5 (Elf5) functions
downstream of the prolactin receptor signaling pathway
and plays an important role in mammary gland develop-
ment. Using conditional mouse knockouts, we have previ-
ously shown that Elf5-null mammary glands exhibit a
complete failure of alveologenesis during pregnancy. The
Elf5-null developmental phenotype is mediated through
alteration in the expression of several critical genes
involved in alveologenesis, particularly those belonging to
the JAK/STAT pathway. Here, we demonstrate that in
addition to regulating terminal differentiation of alveolar
cells, Elf5 also plays a critical role in determining cell fate
and in regulating the stem/progenitor function of the
mammary epithelium. Targeted deletion of Elf5 in the
mammary glands leads to accumulation of cell types with
dual luminal/basal properties such as coexpression of K8
and K14 and an increase in CD611luminal progenitor
population during pregnancy. Further interrogation sug-
gests that the abnormal increase in K141K81cells may
represent the CD611luminal progenitors blocked in dif-
ferentiation. Remarkably, Elf5 deficiency in mammary ep-
ithelium also triggers an increase of adult mammary stem
activity as evidenced by the accumulation of mammary
stem cell (MaSC)-enriched cell population in both preg-
nant and virgin mice and further confirmed by mammo-
sphere and transplantation assays. Additional support for
this phenotype comes from the enriched MaSC gene signa-
ture based on transcriptomic analysis of the Elf5-null
mammary gland. Finally, our biochemical studies suggest
that Elf5 loss leads to hyperactivation of the Notch signal-
ing pathway, which might constitute in part, the underly-
ing molecular mechanism for the altered cell lineage
decisions in Elf5-null mammary epithelial cells. STEM
Disclosure of potential conflicts of interest is found at the end of this article.
The adult mammary gland is an epithelial-rich organ that
undergoes remarkable structural and physiological changes
during pregnancy. The main structures that are primarily
affected by these dynamic changes at this stage are the milk-
producing alveoli and the elaborate ductal network. The epi-
thelial lining of the alveoli and the ducts comprises two major
cell types: luminal and basal [1, 2]. The generation of the
mature and functional lobuloalveolar units of the mammary
gland during pregnancy is governed by endocrine signals gen-
erated predominantly by prolactin and steroid hormones such
as progesterone and a network of critical transcription factors
that activate target genes involved in cell-fate determination,
proliferation, and differentiation [3–5]. One such transcription
factor is E74-like factor 5 (Elf5, also known as ESE-2).
Elf5 is highly expressed in tissues rich in glandular or se-
cretory epithelial cells such as mammary gland, salivary
gland, kidney, lung, and stomach [6–10]. In addition, during
early embryonic development, Elf5 expression is restricted to
the extraembryonic ectoderm, which is essential for mamma-
lian placental formation and the survival of the embryo in
utero [11]. Elf5 is a critical transcription factor at this stage,
since Elf5-null embryos die early during embryogenesis and
exhibit defects in the formation of extraembryonic ectoderm
[6, 11, 12]. Indeed, recent studies have shown that Elf5 is im-
portant for the maintenance of the trophoblast stem cell popu-
lation and is differentially and epigenetically regulated to dic-
tate embryonic and trophoblast cell lineage choices [13].
In mammary glands, Elf5 is highly induced during preg-
nancy and is primarily restricted to the luminal epithelial cells
[12, 14, 15]. The important role of Elf5 in mammary gland
development is clearly evident in the lactation failure and
blocked alveolar morphogenesis phenotype in Elf5 females,
even under heterozygous conditions [12, 16]. The importance
of Elf5 in lactation is further exemplified by the precocious
alveolardifferentiationphenotypeobserved in Elf5-
Author contributions: R.C.: conception, design, collection of data, analysis, and manuscript writing; Y.W.: technical support and
analysis; R.R.: technical support; C.D.: collection of data; Y.K.: manuscript writing; S.S.: conception, analysis, and manuscript writing.
Correspondence: Rumela Chakrabarti, Ph.D., Department of Molecular Biology, Princeton University, Princeton, New Jersey 08554,
USA. Telephone: 330-389-1943; Fax: 609-258-2340; e-mail: firstname.lastname@example.com; or Satrajit Sinha, Ph.D., Department of
Biochemistry, State University of New York, Center of Excellence in Bioinformatics and Life Sciences, 701 Ellicott Street, Buffalo,
New York 14203, USA. Telephone: 716-881-7994; Fax: 716-849-6655; e-mail: email@example.com
accepted for publication April 3, 2012; first published online in STEM CELLS EXPRESS April 20, 2012. V
2012/$30.00/0 doi: 10.1002/stem.1112
STEM CELLS 2012;30:1496–1508 www.StemCells.com
Received August 25, 2011;
C AlphaMed Press 1066-5099/
Page 2
overexpressing virgin mammary glands [16]. These studies
have firmly established this transcription factor as a master
regulator of the alveolar switch. To overcome the problem of
embryonic lethality of mice lacking Elf5, we have used the
Cre-loxP system to ablate Elf5 specifically in the mammary
epithelium [14]. We have shown that conditional loss of Elf5
in mammary glands leads to a complete failure in lobuloal-
veologenesis and alterations in the PrlR/Janus Kinase2/Signal
transducers and activators of transcription 5a signaling path-
way as well as many other known mediators of the mammary
gland developmental program suggesting a much broader role
of Elf5 [14, 17, 18]. Indeed, recent studies on transcriptional
mediators of stem/progenitor cell differentiation and lineage
control mechanisms of mammary epithelial cells (MECs)
have unearthed the role of key players such as GATA-3 and
to some extent Elf5 in these processes [16, 19, 20].
While animals lacking GATA-3 exhibit morphogenetic
block during mammary placode formation and pubertal mam-
mopoiesis, Elf5-null mammary glands examined so far do not
exhibit any early developmental defects in ductal growth and
morphogenesis [16, 19, 20]. However, pregnant Elf5-null
glands harbor an expanded pool of luminal progenitor cells
suggesting that Elf5 might actively influence the dynamics of
mammary epithelial progenitors and their programmed differ-
entiation [16]. Given the broad expression pattern of Elf5 that
extends beyond the pregnancy/lactation window and the
emerging evidence that Elf5 is a prototypic marker for both
human and mouse luminal progenitor population, it is con-
ceivable that this factor may also influence earlier stages of
the mammary epithelial lineage choices [21].
In this study, we used the Elf5 conditional knockout ani-
mals to further understand the underlying molecular mecha-
nism by which Elf5 dictates cell-fate determination and line-
age choices in mammary gland development. Our data show
that loss of Elf5 leads to an unexpected and dramatic increase
in K14þ/p63þ/K8þmixed population of cells in the luminal
compartment, suggesting an increased progenitor cell popula-
tion. Flow cytometric analysis also revealed an increase in
CD61þcells during pregnancy in agreement with previous
studies [16]. Interestingly, sorted CD61þ
showed an increase of double-positive K14þ/K8þcells in
Elf5-null MECs, suggesting that the CD61þfraction might
represent cells with dual characteristics that are blocked in
differentiation toward mature luminal cells. Furthermore, we
also observed an increase of mammary stem cells (MaSCs) in
the absence of Elf5 that is detectable during pregnancy as
well as in the virgin stage. Flow cytometric analyses as well
as mammosphere assays and in vivo transplantation experi-
ments confirmed the increased stem cell activity in Elf5-null
mammary glands in both pregnant and virgin stage. Finally,
our findings that loss of Elf5 leads to hyperactivation of
Notch signaling and that inhibition of Notch signaling can
partly restore the increased stem/progenitor cell activity of
Elf5-null MECs provide a novel mechanistic link between
Elf5 and Notch signaling pathway in mammary gland
Animal Studies
The Elf5-null mice have been described previously [14]. Mice
of C57BL/6 strain (6–8 generations) were used for experi-
ments with virgin mammary glands. The genotype of the con-
trol wild-type (WT) animals was either K14-Cre/Elf5þ/þor
Elf5f/f, whereas the genotype of the Elf5 conditional knock-
outs (Elf5-null) was K14-Cre/Elf5f/f. Animal procedures were
conducted in compliance with the guidelines of the IACUC
Committee of the State University of New York at Buffalo.
Histological Analysis, Immunostaining,
and Western Blots
For histological analysis, mammary gland specimens were
fixed as described in [14]. Antibodies dilutions used for im-
munofluorescence and immunohistochemistry are described in
Supporting Information Table S1. Protein extracts were pre-
pared and Western blot was performed as described in [14].
Antibodies used for Western blot are described in Supporting
Information Table S1.
Primary MEC Culture and Viral Infection
Isolation and adenoviral transduction of primary MECs were
performed as described previously [14, 22]. For lentiviral
mediated knockdown studies, shRNA knockdown vector for
murine Rbpjk/Cbf-1 (TRCN0000097287-88) was purchased
from Open Biosystems Inc. (Huntsville, AL, https://www.
openbiosystems.com/). Control plasmid containing empty vec-
tor was purchased from Sigma-Aldrich (St Louis, MO, http://
were packaged into virus using HEK293-T-cells as packaging
cell lines using helper plasmids VSVG and dR8.9 using stand-
ard protocols. Primary cells were spin infected with virus-con-
taining media supplemented with 2 lg/ml polybrene for 2
hours at 1,000g at 4?C and then either plated in matrigel or
Flow Cytometry/Fluorescence-Activated Cell Sorting
Single MECs suspension was pelleted and resuspended in flu-
orescence-activated cell sorting (FACS) buffer (1% BSA in
PBS). Briefly, MECs were blocked with anti-CD16/CD32 Fcc
III/II receptor antibody (BD Pharmingen, San Jose, California,
http://www.bdbiosciences.com/home.jsp) and stained with a
combination of either (FITC-CD45, FITC-Ter119, FITC-
CD31, CD24-PE, CD29-APC, and biotinylated-CD61) or (bio-
CD24-PE, CD29-FITC, and CD61-APC) for 20 minutes on
ice. FACS analysis was performed using the LSRII Flow Cy-
tometer (BD Biosciences) and data were analyzed using
FlowJo software(TreeStar,
described above was repeated with five or more independent
samples obtained from mammary glands number 4 and 5 of
WT and Elf5-null females at P17.5d and 7-week virgin. Flow
sorting was performed using FACS Aria (BD Biosciences) or
FACS Vantage SE w/DiVa (BD Biosciences).
Inc.). The FACSanalysis
Mammosphere and Colony-Forming Assay
For mammosphere and colony-forming assays, primary MECs
isolated from mammary glands number 4 and 5 of WT and
Elf5-null mice (P17.5d and 6–8 week virgin) were either
plated onto six-well, ultralow attachment plates at a density
of 10,000 cells per milliliter or in matrigel at a density of
1,000 or 20,000 cells, respectively. Cells were grown as
described previously [23, 24]
Limiting Dilution Assay and Extreme
Limiting Dilution Assay
Single-cell suspension of primary MECs from WT and Elf5-
null mammary glands at 6–8 weeks were sorted in combina-
tion with the Lin?, CD24þ, and CD29himarkers to obtain
MaSCs (Lin?CD24þCD29hi) that were then injected into
cleared mammary fat pads. The outgrowths were analyzed at
8 weeks post-transplantation. Transplantation was performed
with cells resuspended in 50% Matrigel and 50% PBS. Fre-
quency of MaSCs in transplanted cell suspension was
Chakrabarti, Wei, Romano et al.
Page 3
calculated using L-calc software (StemCell Technologies,
Vancouver, Canada, http://www/stemcell.com/) or extreme
limiting dilution assay (ELDA) [25]. Single-hit model was
also tested using ELDA and value of slope was 1. MaSC
abundances were assumed to follow a Poisson distribution in
LDAs, and generalized linear models using a log-log link
function were used to derive repopulation frequency parame-
ters. Differences in repopulation frequencies (defined as the
number of cells required for repopulation to be observed 63%
of the time) were determined by relying on the asymptotic
normality of the maximum likelihood estimates.
Real-Time PCR Analyses
Total RNAs were isolated and real-time PCR was performed
as described previously [14]. The gene-specific primer sets
are listed in Supporting Information Table S2.
Gene-Set Enrichment Analysis
We used GSEA v2.0 to perform the gene-set enrichment anal-
ysis (GSEA) on various functional and/or characteristic gene
signatures [26, 27]. Normalized microarray expression data
were rank-ordered by differential expression between Elf5-
null versus WT, using the provided ratio of classes (i.e., fold
change) metric. The details of the microarray experiments
will be described in a separate manuscript. Gene sets were ei-
ther obtained from the MSigDB database v3.0 (SEPT 2010
release) or manually generated. Milk proteins contain a list of
nine genes: Adfp, Btn1a1, Lalba, muc1, Xdh, Wap, Csnkappa,
Csn1s1, and Csnk1g2. ''CDK inhibitors'' include all eight
gene family members (Cdkn1a through Cdkn3); MaSC-enrich
and luminal signatures are significantly upregulated genes (p
< .05 and FC >3) in MaSC-enriched and luminal subpopula-
tions from MECs of virgin mice, which were derived from
microarray dataset GSE22446.
Inappropriate Expression of Basal Markers
and Expansion of K141/K81Dual-Positive Cells
in Elf5-Null Mammary Glands
Previously, we have shown that mammary epithelium-specific
deletion of Elf5 leads to a complete block in alveologenesis
during lactation [14]. To further probe the effects of loss of
Elf5 during different stages of pregnancy, we compared mam-
mary glands from K14-Cre/Elf5f/f(Elf5-null) animals and
their K14-Cre/Elf5þ/þ(WT) type littermate controls. Both
whole mount carmine staining and histological examination of
mammary gland sections demonstrated that at P4.5 there were
discernable morphological alterations including less tertiary
branching in Elf5-null mammary epithelium (Supporting In-
formation Fig. S1). Notably, these effects were more pro-
nounced at later stages of pregnancy in agreement with higher
expression levels of Elf5 at this stage (Supporting Information
Fig. S1, S2 and [12, 15]). Immunohistochemical staining con-
firmed the complete absence of Elf5 protein at all stages of
development in the Elf5-null mice (Supporting Information
Fig. S2). To further characterize the cell population in the
Elf5-null epithelium, we used a panel of basal and luminal
cell markers. While K14þstaining was predominantly re-
stricted to myoepithelial cells of the basal compartment in
WT (Fig. 1C, 1I, 1O), in Elf5-null tissues, there was wide-
spread mis-expression of K14 within the cells of the luminal
compartment (Fig. 1F, 1L, 1R). This distinctive altered
expression pattern was clearly evident at early stages of preg-
nancy (yellow arrows, P4.5) and progressively increased dur-
ing later stages of pregnancy and at parturition (Fig. 1L, 1R,
1X and graph below [WT ¼ 2% vs. Elf5-null ¼ ?50%]).
Importantly, the altered K14 expression in luminal cells was
also detectable in 12 weeks virgin mammary glands (Support-
ing Information Fig. S3). However, in contrast, K5 expression
was restricted to myoepithelial cells in both Elf5-null and WT
type mammary glands (data not shown). This was not surpris-
ing, given the reported asynchronous distribution of K5 and
K14 during mammary gland development [28, 29]. On the
other hand, immunostaining with p63, another basal cell
marker, showed results similar to K14. Indeed, p63þcells
were scattered throughout the luminal compartment in Elf5-
null mammary epithelium (Supporting Information Fig. S4B,
S4D, S4F) in comparison to the myoepithelial-restricted
expression observed in WT, at multiple stages of development
(Supporting Information Fig. S4A, S4C, S4E). These data are
in agreement with Western blot analysis that demonstrated
increase in basal markers p63 and K14 expression in Elf5-
null tissue (Supporting Information Fig. S5). To further exam-
ine the nature of the K14þ/p63þcell types, we performed
dual staining of K8 and K14. K8þcells were uniformly pres-
ent within the luminal layer of control ducts, as expected, and
were surrounded by a single layer of K14þmyoepithelial
cells along the ducts (Fig. 2C). In contrast, in affected areas
of Elf5-null tissue, there was a robust increase of dual K14þ/
K8þpopulation (WT ¼ 0.5% vs. Elf5-null ¼ 44%) (white
arrows, Fig. 2F). This observation was further supported by
increased colocalization of p63 and K8 in the luminal com-
partment in P17.5 mammary glands of Elf5-null (Fig. 2L) in
comparison to WT (Fig. 2I) (WT ¼ 1.2% vs. Elf5-null ¼
16%). Next, we examined expression of smooth muscle actin
(SMA), a marker of mature differentiated myoepithelial cells.
While the outer layer of myoepithelial cells in the Elf5-null
and control mammary glands displayed the normal pattern of
SMA staining, the K14þpopulation in the luminal compart-
ment of the Elf5-null mammary glands was negative for
SMA, suggesting that these mixed-phenotypic cells were un-
differentiated (Fig. 2O, 2R).
Loss of Elf5 Alters Stem Cell/Progenitor
Populations During Pregnancy
Given the prevailing notion that both the myoepithelial and
luminal cell types are descendent of a common progenitor
population that expresses dual K14þ/K8þmarkers, we won-
dered if the stem/progenitor cells were affected in Elf5-null
mammary glands. To test this, we dissected mammary glands
from P17.5d mice and analyzed them by flow cytometry (Sup-
porting Information Fig. S6A) with established surface
markers [24, 30]. We observed a consistent increase in
Lin?CD24þCD29hi(MaSC enriched) cell population (WT ¼
35% vs. Elf5-null ¼ 59.1%) and a decrease in the
Lin?CD24þCD29lopopulation (WT ¼ 32.5% vs. Elf5-null ¼
20.5%) in Elf5-null MECs compared to WT (Fig. 3A, Sup-
porting Information S7A, S7B). The Lin?CD24þCD29locom-
partment can be further subdivided into CD61þprogenitor
cells and CD61?differentiated mature cells [19]. Further
characterization of the luminal progenitors using the CD61
marker showedsignificant
Lin?CD24þCD29loCD61þpopulation in the Elf5-null mam-
mary epithelium in comparison to WT control (Fig. 3B), simi-
lar to earlier studies [16]. The purity of the various sorted
populations was confirmed by real-time RT-PCR (qRT-PCR)
for K14 and K18 as shown in Supporting Information Figures
S8A and S8B. We next performed dual immunofluorescence
staining of the Lin?CD24þCD29loCD61þpopulation for K14
Elf5 Regulates Cell Fate Via Notch
Page 4
and K8 expression. This showed that the CD61þElf5-null
MECs from P17.5 consists of a significantly larger (Elf5-null
¼ 42% vs. WT ¼ 16%) fraction of K14þ/K8þcell population
compared to the WT (double-positive cells are marked by yel-
low arrowheads in Fig. 3C and bar graph in Fig. 3D). Taken
together, these data suggest that in the conditional Elf5
knockout mammary glands, the alveolar differentiation block
is also accompanied by accumulation of premature cells that
might represent stem/progenitor fractions.
We next examined the functional effects of loss of Elf5
on stem/progenitor population proliferation and expansion by
mammosphere and colony-forming assays. MECs derived
from Elf5-null P17.5 mammary gland showed an increase in
primary mammosphere-forming activity compared to WT
cells (Fig. 3F), indicating increased stem or progenitor cell
function. In addition, the mammosphere diameters were larger
in Elf5-null samples in comparison to the WT (Fig. 3E, 3F).
Primary mammosphere-derived cells can initiate secondary
mammospheres, as well as differentiate to various breast line-
ages, thus exhibiting two fundamental properties of stem
cells—self-renewal and multilineage differentiation [31]. To
examine self-renewal and intrinsic stem cell activity, second-
generation mammospheres were counted, which revealed a
distinct increase in secondary mammosphere-forming activity
Figure 1.
was expressed exclusively at the basal cells. Occasional K14 cells were present in luminal compartment as shown by yellow arrow (C). However,
in P4.5 Elf5-null tissue, cells were frequently observed in luminal compartment that were positive for K14 (yellow arrows in F). At later time
points during pregnancy and lactation (P12.5d-L1), K14 was expressed exclusively at the basal cells as in (I), (O), and (U) in the WT tissue. In
Elf5-null tissues, large numbers of K14-positive cells were observed in the luminal compartment of the mammary epithelium (L, R). Yellow
arrows indicate aberrant localization of K14 in luminal compartment and red arrows indicate the unaffected normal luminal cells negative for
K14 at term (X). Bar graph below represents quantification of percentage K14þpopulation in the luminal compartment. Data represent mean 6
SD of average of 10 image fields from six animals per genotype, p values calculated by t test for statistical significance. Abbreviations: Elf5,
E74-like factor 5; WT, wild-type.
Altered expression of K14 during pregnancy. (A–F): P4.5, (G–L) P12.5, (M–R) P17.5, and (S–X) L1. At P4.5d, in WT tissues, K14
Chakrabarti, Wei, Romano et al.
Page 5
in Elf5-null MECs compared to the WT cells (Fig. 3G).
When individual mammospheres were plated on collagen-
coated chamber slides, most cells in the spheres differentiated
and migrated out of the center after 4 days of culture. Immu-
nostaining of differentiated mammospheres identified K8þ
and K14þcells from both WT and Elf5-null mammospheres
(Fig. 3H), confirming that the spheres were capable of differ-
entiating into both luminal and basal lineages. Of note, simi-
lar to Elf5-null mammary glands in vivo, an increase in dual-
positive (K14þ/K8þ) cells was also observed in the differenti-
ated cells of the Elf5-null mammospheres (Fig. 3H). We next
compared the colony-forming capacity/progenitor activity of
WT and Elf5-null MECs that were grown at clonal density in
Matrigel and examined their potential to form spherical colo-
nies. Elf5-null MECs formed more spherical colonies in com-
parison to WT per 1,000 MECs seeded (Fig. 3I, 3J). Average
sphere size in Elf5-null was larger than WT; however, this
increase was not statistically significant (data not shown).
These results were further confirmed by examining mammo-
sphere formation and colony-forming ability of purified sorted
subpopulations fromWT
and Lin?CD24þCD29lo), respectively
(Supporting Information Fig. S8C, S8D).
and Elf5-nullMECs,
Increased Stem/Progenitor Cell Population
and Activity in Elf5-Null MECs Obtained
from Virgin Animals
We next examined whether loss of Elf5 also affects stem/pro-
genitor cell population in virgin animals. Flow cytometry
Figure 2.
of K14 and p63 is present exclusively in basal compartment in WT tissue (C, I). However, in Elf5-null tissue, an increase in double-positive
K14þ/K8þwas observed as shown by white arrows (F). In some areas, almost all cells in mammary epithelium were positive for K14 and K8,
particularly at parturition as shown by white arrows. (L) Increase in K8þ/p63þdual-positive cells was observed in Elf5-null mammary gland
(white arrows in L). K14þcells in the luminal compartment are negative for smooth muscle actin (SMA) (O and R). Altered localization of K14
was observed in the luminal compartment (columnar cells) in Elf5-null mammary epithelium (R) in comparison to WT tissues, where K14 was
expressed predominantly at the basal cells (O). Note: SMA staining is absent in the luminal compartment and is present only in the basal cells in
both WT and Elf5-null mammary epithelium. Bar graph below represents quantification of percentage K14þ/K8þpopulation (left) and p63þ/K8þ
population (right) in the mammary epithelium. Data represent mean 6 SD of average of six images from 4 to 6 animals per genotype, p values
calculated by t test for statistical significance. Abbreviations: Elf5, E74-like factor 5; WT, wild-type.
Colocalization of K14/p63 and K8 in luminal compartment during pregnancy. (A–F): L1, (G–L) P17.5, and (M–R) L1. Expression
Elf5 Regulates Cell Fate Via Notch


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text:
I am a piece of trash. No seriously. I feel so guilty for taking so long. There isn't really a good excuse. Uni was crazy and I hade like 300 exams and I still have essays to write and exercise sheets to fill out and it narrowed down the time I had for writing fanfics. Ughhh.
But I finally finished up a new chapter and I wish it could have been longer than that but I wanted it to end there. But I PROMISE that the next chapter will come shortly, as it is almost finished up anyways. Also, I am almost sad to see this fic's end coming up soon. It's weird because this is my first RivaMika fic every and it's like a little child for me.
Of course I will still continue Blutrunst and I already have an idea for a new fic after this one is finished. There I can also include the newest revelations of the manga! I was actually quite excited by Levi's last name tbh. (No, I don't think they are related at all. Really, I don't.) What did you think of the latest chapters?
Anyways, let's stop rambling. I hope you like this chapter!
Disclaimer: Nah, still not owning anything here.
Mikasa was putting on a shirt – one of Levi's which was a little too tight for her – as someone knocked at the door. Instinctively, Mikasa turned her back to it, as Levi went to get it. It wasn't necessary, though because waiting on the other side was Armin.
As the door was opened for him, his bright blue eyes travelled past Levi, eying the room for his childhood friend and a relieved sigh escaped the young man as they found Mikasa.
Levi wordlessly stepped aside to let him in and Armin's feet brought him directly to her. Mikasa turned around to embrace him and at first Armin's arms tightly locked themselves around her. But as she let out a little gasp – trying to disguise her pain – his embrace loosened and he leaned back to look at her.
They shared a warm smile and Levi, standing a little to the side – felt out of place, next to their all so touching reunion.
"I'm glad you're okay." Armin's arms let go of her and he stepped back, still that smile plastered on his face. Mikasa's however, was gone as if it had only been a small notion of emotion. She just nodded and her arms enveloped her own body, avoiding Armin's gaze. He drew his eyebrows together, sensing her change in mood.
"You are okay, aren't you?" This time his eyes shifted towards Levi, who was leaning against the wall to their right, watching them with pretend disinterest. His eyes also locked with Armin's and he didn't show any reaction to his questioning look, just stared back at him, until the blond one wavered beneath it.
"I am fine," Mikasa chimed in, breaking Armin's and Levi's eye contact.
"The cat it out of the bag, though… obviously." Armin very well knew what she was referring to as he had already deducted the current circumstances. It hadn't been a hard puzzle. Mikasa was standing here as a woman after all and not as a disguised one at that.
"And what now?" Armin nervously shuffled on his feet, looking back and forth between Levi and Mikasa, as they shared a silent conversation of looks now.
"She will leave, that's now." Levi pushed himself off the wall, walking towards them in a quick pace. Mikasa turned her head to him, her face wearing an annoyed expression, her tongue clicking.
"I thought we talked about that."
"Yes, and I decided that we won't stick to your bullshit plan."
Armin stepped back, sensing a conflict arising before him. The blond didn't know whether to stop Mikasa or to just leave them be. He didn't really looked forward to be a casualty on a battlefield. Not this one, that is.
"I explained to you why I have to stay. I cannot leave. I just can't." Mikasa's tone seemed final and she fully turned to the Corporal now, hovering over him. But she really didn't intimidate Levi; not in the slightest. His blue eyes turned into an icy storm, giving out a dangerous vibe and Armin wondered if that was the look he wore to slice monsters.
"Listen closely, Mikasa, right now I am supposed to kill you," he moved forward, making Mikasa to step back.
"But if someone else finds out about your "special circumstances", then there is no guarantee." Levi cornered the young woman in front of him. Her legs touched the bed now and with another final step of Levi, she was sitting on it and now it was him to hover. She sat there and looked up to him but if his eyes were icy storms, then hers were wildfires.
"I don't care for my life. I care for my family," she said in a low voice, dipped in the sweetest poison one could die of. Levi stared at her and it was clear that he wanted to say something, wanted to yell at her because she made him boil like no one before.
I care, lingered in his mouth. But it was too disgustingly sweet to spit out, so he swallowed her poison and said: "Then do what you want. I won't care." instead.
Armin and Mikasa walked back to their dorm in silence. After that heated confrontation, Mikasa took the medicine Hanji had left behind and they both walked out of Levi's room – without another word. As she looked down to the ointment in her hands, she tried not to think of those hands that had traced her marks with it on it. It was hard not to think of him or the whole situation at all, when she was wearing his tight shirt that smelled of lime and when those rough palms still echoed on her skin. Her lips were still dry from the contact with his and she could swear she could still taste it. The kiss.
Oh damn it all.
As they arrived at their room, Mikasa was glad to find it empty. Connie was probably somewhere else. Maybe he was hurt, too. Maybe he didn't make it. It almost shocked Mikasa how little she cared right now. She should care. She really, really should. But no matter how hard she tried to think of all her other comrades, she couldn't find the will to worry for them, too. Her head was drowning in problems and anger and worry and the smell of lime and medicine. Her family, Eren, was in danger. He turned into a Titan. He controlled it. The fact that she couldn't run down into the dungeons and to see him, swallowed her up like a Titan's mouth. Also, she was in danger, too. With her wounds from the battle, there was no way she could bind away her breasts as tightly as usual. Just the thought of it made her dizzy.
Mikasa laid in her bed and closed her eyes and even though everything seemed to be a mess right now, she fell asleep the moment her lids hit her eyes and the dreamt of monsters and lemon trees.
Levi stared at the new bed sheets he put on right after his guests left. The used ones were clutched in his hands and he had to fight the ridiculous urge to bring it to his nose. Just the thought made him cringe and he felt stupid for even thinking about it. If only for a second. He threw the sheets in the basket he had for his laundry instead, with a little too much force.
His room felt weirdly empty now, after all the excitement, and the stress and all the new revelations. Levi was surprised to find himself not totally exhausted, though. He should be collapsing onto his bed right now, enjoying the silent moments he had for now. Who knew how long they would last until an Abnormal's foot would crash yet another wall?
Instead, he was standing here in his room, trying to push back thoughts of one particular person that made his day even more fucked up than before. He was so angry. Angry at her and himself, at the government and at Titans.
Not wanting to stay in his chambers for another second, he almost stormed out and followed the stone halls down to the dungeons.
No one was left here apart from two guards, which he had to ask twice until they left.
Eren sat up as the Corporal pulled a chair closer to his cell, the only occupied one between lines of silent echoing spaces.
They stared at each other for some minutes, Eren anxious and Levi deep in thought, until:
"I just met your sister, Eren." Levi leaned back, crossing his arms and watching Eren's reaction. His words were like a trigger to the boy, who immediately moved. He stood up from his bed and ran towards Levi, not coming all that far, though, due to the chains around his wrists.
"What did you do to her? Where is she? What the hell happened?"
Levi watched how the young man in front of him filled up with anger and concern, his eyes wide and arms pulling. Metallic sounds echoed through the basement from Eren's rattling.
Levi didn't talk right away. He didn't know why he was doing this. Why he was even here. Perhaps he couldn't stand to stay in his room right now and because his head was filled with all kinds of problems he never imagined to face.
"Where is she?!"
The Corporal's eyes snapped up to him, eying him for yet another second. Why was he being so cruel right now? Who knew, it could be that he was getting a kick out of this.
"Relax, she is fine. Probably sleeping. She was wounded in battle but it's nothing serious."
His words finally calmed Eren down and his arms fell to his sides, his face downwards.
Levi heard his heavy breaths that held relief in them but soon his face moved up again and this time an expression of desperation clothed his young features.
"Please, don't hurt her. I beg of you." Eren tried to bring his hand forward but the chains worked their magic again and Eren quickly gave up on that idea.
"It was my fault, she-she did it for me."
Levi stood up from his chair and started to walk around the small and dark space. He didn't look at Jaeger, as he – yet again – waited a little too long to answer. As if he needed to think about it. There was nothing to think about. And yet…
"Tell me, Eren," he turned to the young man again, "do you really think you are in a position to make demands? And one at that, too." He turned around. "Asking me to break the law?"
His deep voice was ice cold and he wondered why he was torturing the boy like that. He was being cruel and right now selfishly venting his anger. But Levi was never a nice person, after all.
"Please, Corporal. I would do anything. Give anything I have. She is my family."
Levi had a bitter taste in his mouth as he listened to Eren's shaking voice, begging him. This wasn't delicious or releasing in any way. It made him sick. Sick of himself. And it made him even sicker to wonder what she would think of what he did just now. She made him sick, feverish even, and he doubted there was a medicine to cure him from her.
"Go to bed, Eren and don't break you head over things that need not fucking worry to waste one."
And with that he left him behind.
Mikasa felt disoriented when she opened her eyes. She expected to see sunshine falling through the window but the sky was dark as night and she slowly sat up and tried to make out her surroundings. Did she sleep through the entire next day? It couldn't be. She usually never slept all that long anyway and she also didn't feel rested enough for it. As her eyes adapted to the darkness in the room, she slowly stood up – ignoring the screaming pain of her ribs and chest – and walked close which hung next to the door of their room. The little light, provided by the room finally supported her theory: She didn't sleep through the day, she only slept for 4 hours. It was still early morning. Too early for the sun to rise.
For a moment, Mikasa contemplated to go outside and clear her mind because she was sure she wouldn't be able to sleep anymore. But she quickly dismissed the idea in fear of meeting someone. A shower, however, was something she couldn't deny herself. No one should be there now after all. After a won battle, normal people earned sleep by their victory. But Mikasa did not win anything yesterday.
She silently gathered a change of clothes, towels and also medicine and fresh bindings as she set off to the shower area. She was careful not to wake Armin – and also Connie who was laying in his bed now, too. She had stopped to look at him in the blackness of the room and he seemed to be fine enough and Mikasa told herself that she was very relieved and happy about that but deep down she knew that she only checked because she felt guilty for not caring enough.
As she walked down the empty halls of the headquarters, she let her thoughts wander away and of course they didn't stay far away from Eren. She longed to see him, maybe just for a second, to see he was alright. It would be more than enough for her. For now.
She didn't know what would happen now but she knew that she wouldn't let anything happen to him. This is why she was here after all.
As she expected, the showers were totally empty. No one in their right mind would get the idea to shower at 3:30 am.
When Mikasa put her stuff on one of the wooden benches, she thought about all the time she actually spent in these rooms, especially on her knees and scrubbing. It had almost become a habit, to come here every evening and be alone for an hour or two – not that she missed the actual part of cleaning anything, really. She really knew to use her time better than that and for once she was able to participate in the dinners and not to eat alone in her room afterwards. (Bless Armin for always remembering her.)
The hot water felt heavenly and she didn't regret her decision to come here. Mikasa found herself standing under the hot steam of water, unmoving and eyes closed for several minutes. It felt good to be able to do nothing, to be alone after everything that had happened. Her mind seemed be unable to relax until now and she knew that, as soon as the water stops falling down on her, she would go back to be tense and worried. She had an unfortunate taste in her mouth and the few hours of sleep she had didn't really do anything good to her. It rather was like she was more exhausted than before. And the worst thing was that she saw no solution to her problems. The only thing she was sure about, was that she would try. She would find a way. She always did.
Eventually, Mikasa had to get out of the shower. Her skin was red and heated up by the stream and as she stepped out of the shower, she felt dizzy. She grabbed her towel, wrapped it around her and sat down for a file. Her head upwards towards the ceiling, her eyes tiredly roamed around the room, thinking of nothing in particular for a while. She counted the tiles for a while, thought about how she never cleaned up there and then felt silly because who on earth did that.
"If this is your idea of being careful, then you really suck at it."
The sharp voice made her jump and she turned her head a little too fast, making the room spin around her. She steadied herself at the wet tiled walls of the shower room as she looked over to Levi, who was standing by the entrance, leaning against the wooden door. His face gave away no emotions, only look at her, travelling down her almost naked body.
Mikasa felt a blush creep up her face and subconsciously, she drew the towels tighter around her, making her wounds pulsate.
Why did he have to be here? Why was he everywhere she went, in her mind and body? It didn't make any of this easier.
Levi pushed himself off the door and walked up to her. Mikasa averted her eyes to avoid his gaze and usually she would have stood up to turn around but she didn't trust her legs to safely hold her in place yet. She really shouldn't have showered for that long.
Eventually, he sat down next to her and his head was turned in her direction, apparently waiting for her to look up to him. She didn't. Instead she focused on his hands on his laps.
"Eren is alright. I visited him a few hours ago. He knows that I know about you." He spoke fast, almost too fast for Mikasa to understand but she did and her head snapped up to his now, sending another wave of dizziness through her body, which she tried to ignore as best as she could.
Now it was Levi that had turned his face from her, staring ahead and not saying anything else even though she waited for more to come. But she understood why he was telling her this and she was grateful. Very grateful even. He being here, with the clear intention to see her and tell her this… he did so much for her even though all she did was lie and fight. She didn't regret it of course. Not one single action of hers was clouded by regret. Disguising herself and being here. Denying to back off and intending to save Eren.
He probably knew it. And he still helped her.
Levi suddenly stood up, not looking back at her and started to retreat. Mikasa's eyed wided as she saw him leave and without thinking about it, she stood up and extracted a hand to stop him.
"Wait," but she didn't come any farer because another wave of spinning came up the minute she stood up and for just a little instance all she saw was black and she felt how her knees gave in.
She should have fallen onto the ground but before she felt them, she knew two hands would grip her and stop her fall. And of course they did.
Her sight was shaky as she made out Levi's face close to hers and his hands were wrapped around her arms, holding her up and in place. They looked each other in the eye for the first time since he came in here and all they did was to keep staring at each other. Until Mikasa's eyes snapped down to his lips and she saw them getting closer. She felt him exhale very slowly and his hands tightened their grip around her arms to the point of bruising her but she didn't even feel it. Instead her eyes fluttered shut and she waited for his lips to meet hers.
But they didn't.
Instead she was sitting on the bench again and the Corporal had let go of her, only leaving the memories of him there. He stood in front of her and she looked up to him in question. His hand was covering his mouth, his eyes were closed and she saw how his cheeks were not as pale as the rest of his face.
"Get dressed. We can't have anyone see you like this."
And then he left and Mikasa didn't stand up to stop him this time. Instead she just sat there and didn't listen to his advice for another 20 minutes until she noticed that her body was shaking from the cold, every ounce of heat gone.
An entire week passed, she realized as she carefully applied her bindings. It still hurt and instead of getting better, it felt like it hurt more every day. Armin watched her back as she sat on her bed, not paying attention to him and his depreciative look.
"You won't heal that way," he repeated for the 50th times. At least it felt like that for Mikasa. And still she didn't react to it. Instead she finished binding them around her chest, putting on her shirt and slowly standing up from her bed.
After only two days a new routine developed for the soldiers of Trost. Those who were hurt, were bound to the bed to heal. Everyone that could walk was assigned to walk around the wreck of the city and search for the alive and dead. The Legion was able to capture two Titans and Mikasa heard that they were experimenting on them.
Every day, Hanji came over to check for her wounds and every day he would say the same thing: Leave. But Mikasa wouldn't.
She didn't see him after the incident in the shower. That is, they didn't talk to each other. She saw him each and every day of course but he would totally ignore her and even when she thought that he would maybe glance over to her, she only ever found his back towards her. She tried not to think about it but it was hard.
She still wasn't allowed to see Eren and it frustrated her more than anything else. Sometimes she would space out and Armin had to elbow her several times to pull her back to the here and now. She couldn't allow for this to happen anymore. But of course it did.
She put on her uniform and left the room, left Armin and his pleads to listen to him and she was glad that he didn't follow her.
The young solider went outside to the town, a list of names in her hand and the gear heavy around her hips.
They didn't have any big and official ceremony like the soldiers before them that got to join the Legion. Fully join them not as a recruit but as a private.
As the emerald cloak and the rest of her uniform was handed to her, she tried not to look up to Levi but she failed of course. But he wouldn't look to her. Hanji did, however, and his eyes held worry for her and it made her feel even worse. But she ignored it like everything else and simply put on her cloak and she could swear that the Wings of Freedom burned on her back. Even now, days later and as she walked down those battered streets. Her feet carried her somewhere around and she wasn't even sure if she had checked this part of town already or not. Ruins and stones looked so much alike on dead battlefield. They called it humanity's victory but all Mikasa could see was death.
"You are Masaki, aren't you?"
Mikasa stopped, confused by the voice that spoke the words. She slowly turned around and was surprised to face that person. They stood a little aside in a narrow alley and a little movement of their hand motioned her to come closer. She did of course because she never expected to see them here. Mikasa followed the surprising visitor deeper into the alley until both of them were swallowed up by shadow and they stopped and turned around, letting the grey cloak fall from their face to reveal it whole. But Mikasa had already recognized them, anyway.
"I am glad to have found you so fast. I expect you know who I am," they said and Mikasa was surprised by the firmness of their voice but it made sense.
"Yes," she replied and stepped a little closer to them. "And I expect you know who I am, too… Petra Ral."


text: Kevin C. Cox/Getty Images Sport What Will Green Bay Packers Offense Look Like Without Jordy Nelson? By Sam Tastad August 27, 2015 | ESPN 99.1 The Green Bay Packers lost star wide receiver Jordy Nelson to an ACL injury last week against the Pittsburgh Steelers in the preseason. Read More|Comment Category: Sports Tags: Aaron Rodgers, Green Bay Packers, jordy nelson, NFL Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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